The immune system senses infections (e.g. virus or bacteria) as well as pathologic situations (e.g. necrosis) by the detection of danger signals termed respectively PAMPs (Pathogens-Associated Molecular patterns) and DAMPs (Damage Associated Molecular Patterns). Those molecular signals are detected by several families of specific receptors termed pattern recognition receptors (PRRs). PAMPS can be proteins (e.g. flagellin from bacteria), conjugated lipids such as lipopolysaccharides (LPS), or nucleic acids. In this later family, different forms of mislocalized nucleic acids are recognized by different PRRs present in endosomes: unmethylated CpG motifs in bacterial DNA are detected by Toll Like Receptor (TLR)-9, single stranded RNA (ssRNA) is detected by TLR-7 and TLR-8 while double stranded RNA (dsRNA) is detected by TLR-3. Those receptors are expressed in distinct cell populations (Hornung V. et al., 2002, J. Immunol. 168, 4531-4537 and Jarrossay D. et al., 2001, Eur. J. Immunol. 31, 3388-3393) and trigger different intracellular signals that result in different types of immunostimulation i.e. induction of different types of cell surface activation markers and of different types of cytokines. For example, stimulation through TLR-7 is particularly efficacious to trigger interferon-alpha production while stimulation through TLR-3 is particularly efficacious to trigger interferon-beta production (Sandor F. and Buc, M., 2005, Folia Biologica (Praha) 51, 188-197). Of interest, pathogens and pathological situations can trigger more than one PRR. For example Gram-negative bacteria can stimulate innate immunity through TLR-4 by LPS and also through TLR-9 by DNA. Thereby, several cell types get activated and consequently cumulative immune responses can be triggered. This fact was overlooked and synthetic PAMP formulations developed with the goal of inducing therapeutic immunomodulation to fight chronic diseases such as persistent virus infections or cancer use one single PAMP. Using ssRNA it was recently reported that a clinically acceptable immunostimulating formulation can be obtained by combining in specific conditions (i.e. low salts) the natural cationic peptides termed protamine and ssRNA (Rettig L. et al., 2010, Blood 115, 4533-4541 and WO 2009/144230). This formulation activates TLR-7 and TLR-8 but as expected not TLR-3.
The present invention is based on the observation that in adequate conditions ssRNA and dsRNA can be co-formulated into particles. The immunostimulation obtained by these ssRNA/dsRNA particles is unexpectedly higher than the immunostimulation obtained by particles containing each RNA individually. The production of both interferon-alpha and interferon-beta by human immune cells is higher using the chimeric particles than when using respectively ssRNA- or dsRNA-containing particles. Thus, there is a synergy between the different TLRs and/or different cell types resulting in some kind of immunobiological resonance that is of upmost interest for an immunomodulating drug.